The respiratory epithelium is the first line of defense against inspired pathogenic microorganisms. One component of this defense is the production of antimicrobial peptides such as -defensins and cathelicidins. The long-range goal of our research has been to understand host-pathogen interactions in the airway epithelium. Identification of a non-toxic agent that could increase antibacterial capabilities of the airway through transcriptional mechanisms could have long-reaching implications for the treatment of airway infections. Toward this goal, we recently demonstrated that an antimicrobial peptide, the cathelicidin LL-37, can be induced in airway epithelial cells (AEC) grown in an air-liquid interfac (ALI) by the hormonally active form of vitamin D, 1,25(OH)2D3. In addition, we observed the induction of a novel innate immune receptor, Triggering Receptor Expressed on Myeloid cells (TREM-1) on AEC. After TREM-1 levels are increased by 1,25(OH)2D3, activation of this receptor results in stimulation of innate immune mediators, including -defensins. We have further identified transcription factor binding sites in the promoter of these 1,25(OH)2D3-regulated innate immune genes that may be required for the induction. Thus we have uncovered a novel method for innate immune stimulation of airway cells. Based upon these results, we hypothesize that 1,25(OH)2D3 can increase the antibacterial and overall innate immune response of the airway epithelium. However, in order to address this hypothesis, we must first understand the relationship between vitamin D and innate immunity in the airway. Thus, we are proposing here an exploratory study to characterize the effect of vitamin D on innate immune pathways and on in the innate immune defense of the airway. In specific aim 1 we will characterize the cross- talk between the TLR-based innate immune pathways and the vitamin D pathways regulating LL-37 and TREM-1 expression in vitro. In specific aim 2, we will define the mechanism of innate immune regulation of the vitamin D regulatory pathway in airway epithelial cells through a characterization of the pathways and transcription factors leading to LL-37 and TREM-1 gene expression in airway epithelial cells. In specific aim 3 we will determine the effect of 1,25(OH)2D3 on innate immunity in a mouse model of airway infection, using our existing airway infection model of intranasal inoculation of Streptococcus pneumoniae to quantify the effect of 1,25(OH)2D3 on airway host defense in vivo. Since murine cathelicidin is not regulated by vitamin D, we will examine the effect of 1,25(OH)2D3 in vivo using a humanized mouse expressing the human LL-37 gene under the control of its own promoter. The results of our three aims will provide a solid foundation for further in-depth analysis of the roles played by these innate immune mediators in host defense of the airway, and of the potential for the development of 1,25(OH)2D3 treatment as a therapy for airway infections. This application is appropriate for the R21 mechanism because it involves considerable risk but may lead to a breakthrough in vitamin D and innate immunity that could have a major impact on research in the field of airway infections.